Creatinine analysis

ABSTRACT

FOR REPRODUCIBLE QUANTITATIVE DETERMINATIONS OF CREATININE IN AN OPTIONALLY DEPROTEINIZED BIOLOGICAL FLUID, AN AQUEOUS PICRIC ACID/LITHIUM HYDROXIDE SOLUTION IS SEPARATELY ADMIXED WITH EQUAL VOLUMES OF EACH OF (A) THE OPTIONALLY DEPROTEINIZED BIOLOGICAL FLUID, (B) A STANDARD SOLUTION HAVING A KNOWN CREATININE CONTENT AND (C) A BLANK. THE LIGHT ABSORPTION (EXTINCTION OR TRANSMISSION) IS THEN MEASURED FOR EACH OF THE RESULTING ADMIXTURES. THE CREATININE CONTENT OF SAID OPTIONALLY DEPROTEINIZED BIOLOGICAL FLUID IS READILY CALCULATED FROM THE OBTAINED DATA.

United States Patent 3,557,018 CREATININE ANALYSIS Giinter Scheuerbrandt, Darmstadt, Germany, assignor to v E. Merck A.G., Darmstadt, Germany N Drawing. Filed July 17, 1968, Ser. No. 745,389 Claims priority, application ('ermany, July 20, 1967,

Int. Cl. C07c 79/24; G01 33/16 US. Cl. 252-408 6 Claims ABSTRACT OF THE DISCLOSURE BACKGROUND OF THE INVENTION This invention relates to compositions and methods for the detection of creatinine.

A number of tests have been devised for the detection of creatinine. These include such tests as Jatfes Test, Kerners Test, Salkowskis Test, Thudichums Test and von Maschkes Test.

In practice, however, the predominantly employed method is that described for the first time in Zeitschrift fiir physiologische Chemie [Periodical for Physiological Chemistry], vol. 10, p. 391 (1886) and vol. 41, p. 223 (1904) wherein the creatinine is converted into red creatinine picrate by the addition of sodium picrate in an alkaline medium. In accordance with that process, e.g. a serum deproteinized with picric acid is mixed with solution of sodium hydroxide; thereafter the amount of thus-produced creatinine picrate is quantitatively determined by means colorimetry [Biochemische Zeitschrift (Biochemical Periodical), vol. 291, p. 354 (1937)]. As a variant of that process, the deproteinization is etiected with trichloroacetic acid, the resulting supernatant is successively treated with picric acid and (aq) sodium hydroxide, and the creatinine content of the initial serum is then determined by measuring the extinction at 530-550 m However, the picric acid and the sodium hydroxide solution must be added as two successive separate pipette additions. This necessity of using the pipette twice detracts from the accu racy of the determination and requires, in the usually conducted series of tests, a substantial increase in the time and work involved in the determination. Furthermore, a relatively large quantity of serum is necessary for these conventional creatinine determinations.

Another method [Arztliches Labor (Physicians Laboratory), vol. 11, p. 175 et seq., particularly p. 177 (1960)] recommends a creatinine determination by means of picric acid/sodium hydroxide solution after the serum has been deproteinized with tungstate and sulfuric acid at a pH of 2.1-2.2. However, this process does not yield satisfactory results. The extinction measurement must be conducted in the range of 460-530 mg, especially 480-500 m This range yields high blank values, precluding accurate results. According to this method, it is impossible to obtain a measurement at the wavelength of Hg 546 m which is easily measured with a filter photometer with a mercury lamp (devices which are generally readily available). This conventional method further requires the de- 3,557,018 Patented Jan. 19, 1971 SUMMARY OF THE INVENTION Bearing the above inadequacies of the prior art in mind, it is an object of the invention to provide a color reagent for the qualitative and quantitative determination of creatinine wherein the color reagent should facilitate the quantitative determination and make such determination possible with a minimum amount of specimen to be tested.

Another object is that the color reagent permit light absorption readings which can be accurately and reproducibly translated into creatinine concentrations.

A further object is to provide a color reagent which can be premixed in advance of creatinine determinations; it should be capable of addition to the specimen in a single addition, thus avoiding the possibility of plural measuring errors and reducing the overall time required.

A still further object is to provide a reagent which can be metered and employed in dry form.

An additional object is to provide a process to measure creatinine concentration, particularly without diluting a specimen with water, as required by conventional methods.

Upon further study of the specification and claims, other objects and advantages of the present invention will become apparent.

T o attain the above objects, a color reagent comprising lithium picrate and lithium hydroxide is employed for the quantitative determination of creatinine by measuring light absorption of formed creatinine picrate. The color reagent makes it possible to determine the creatinine content of a fluid within a short period, e.g. 8-10 minutes, after adding said reagent to the fluid. Only a small sample of fluid, e.g. 0.5 milliliter (ml.) of serum, is required for an accurate creatinine determination with the reagent. Moreover the light absorption can be measured at any desired wavelength between 500 and 550 millimicrons (ma), especially at the favorably positioned wavelength of 546 m (measured by conventional filter photometers with a mercury lamp).

The accuracy of creatinine determinations with said color reagent is excellent. A reliable, simply manipulatable reagent has thus been created for the determination of creatinine; it yields results which can be accurately reproduced and is particularly suitable for series tests.

DETAILED DISCUSSION OF THE INVENTION The color reagent of this invention is particularly suitable for the determination of creatinine in biological fluids, such as serum, blood, plasma, urine and cerebrospinal fluid, as Well as for samples of foodstuffs and feed, such as meat extract, which can contain creatinine. Likewise, the determination of synthetic creatinine required, in particular, for the preparation of standard solutions for the creatinine determination in biological fluids can be conducted readily and accurately with said color reagent.

Biological fluids evaluated for creatinine content are first, if required, deproteinized in a conventional manner by means of a strongly acidic compound. Trichloroacetic acid and perchloric acid are especially suitable for deproteinization. Trichloroacetic acid is preferred. The

acidic compound precipitates protein; thus-precipitated protein is removed by centrifugation or another conventional separation method from biological fluids.

The color reagent can be present in a liquid solution or in the solid phase. Advantageously, the light absorption of the creatinine picrate (formed by the reaction of said color reagent with creatinine in the fluid being analyzed) is measured in a pH range of about 9-44, preferably at a pH value of about 12. Accordingly, it is suitable to adjust the pH value of the alkaline lithium picrate solution (the color reagent) so that, after the deproteinizing agent has been neutralized, there is sufficient excess alkali present to obtain the above-mentioned favorable pH range during the color reaction. In general, the weight ratio of lithium picrate to lithium hydroxide is 03 to 0.811, preferably 0.55 to 0.65:1, respectively. I

Advantageously, a solution of the color reagent is prepared from a picric acid solution and an excess of a lithium hydroxide solution. In this connection, an aqueous picric acid solution containing at least about 0.5%, H

i.e. 0.5 gram (g.) per 100 g. of solution, of picric acid is suitably employed. Preferably, a saturated (at room temperature, i.e. 20 C.) picric acid solution (picric acid content approximately 1.2%) is used. A picric acid solution adjusted to a weakly acidic pH by the incorporation therein of lithium hydroxide is of special advantage. In such a solution, the picric acid is present, in part, as lithium picrate. Even upon cooling, for example, to temperatures of about (3., picric acid does not crystallize out of such a solution. In this manner, a stable picric acid solution of a higher concentration, e.g. 3%, can be produced.

The lithium hydroxide solution employed advantageously contains about 8-12%, preferably about 10%, lithium hydroxide.

The color reagent is obtained in ready-for-use form by mixing the picric acid solution, which optionally contains lithium hydroxide, with the lithium hydroxide solution. In a preferred embodiment, 1.2% picric acid solution is mixed with a 10% lithium hydroxide solution. In this preferred embodiment, a volumetric ratio of picric acid solution/lithium hydroxide solution of 3:1 to 6:1, especially about 5: 1, is favorable.

Said color reagent should be prepared within a short time prior to use. Most advantageously, the several solutions are mixed immediately before use. However, it is possible to store the thus-prepared mixed reagent for as long as 24 hours before use.

In some cases, the aqueous solution of the reagent can also contain an inert organic solvent miscible with water, such as ethanol. However, the addition of such an organic solvent generally does not afford any special advantage.

Ready-for-use solution of the components of the color reagent is alternatively prepared, for example, by adding lithium hydroxide in solid form to a solution of picric acid.

The ready-for-use solution of the color reagent can also be freeze-dried in a conventional manner for increasing the stability thereof. Such a solid form of the color reagent need merely be dissolved in the required amount of water before it is used.

A freeze-dried solid preparation of the color reagent has advantages over a mixture of solid picric acid and solid lithium hydroxide because material which might give rise to turbidity after the addition of water could be filtered 01f before freeze drying.

The color reagent can also be prepared in solid form by premixing the solid picric acid and the solid lithium hydroxide. Due to the explosiveness of picric acid and picrates, special precautionary measures must be employed, however. It is recommended that the picric acid be mixed with inert diluents, as is customary in conjunction with explosive substances. Preferred diluents are water-soluble inert salts, such as water-soluble alkali salts, e.g. lithium, sodium and potassium halogenides, such as lithium chloride, bromide or iodide, sodium chloride, potassium chloride, or suitable water-soluble sulfates, e.g. sodium sulfate.

Insofar as the color reagent of this invention is employed in the dry state, e.g. also as a freeze-dried solution, the components can be selected so that a metered dosage of the reagent is exactly sufficient for one individual determination. It is then only necessary to add a dosage of the color reagent during each creatinine evaluation. In such a case, the absolute value would be 1 to 50 mg. preferably '20 mg. lithium picrate and 3 to mg. preferably 33 mg. lithium hydroxide.

'For determining the concentration of creatinine in any fluid sample, the color reagent is added to a fixed amount of the sample (previously deproteinized if it initially contained protein). After an incubation period of from about 5-20 minutes, preferably about 8-12 minutes, the light absorption (extinction or transmission) of the thusformed creatinine picrate is measured. Preferably, the extinction is measured advantageously in a range between 500 and 550 me, for example, at 520 m by means of a spectrophotometer, at 546 m by means of a filter photometer with a mercury lamp, or at 509 me by means of a filter photometer with a cadmium lamp. The sample should be maintained at about l0-30 C., preferably at about 20-25 C. during the evaluation. The measured light absorption is proportional to the amount of creatinine contained in the sample.

Determinations of creatinine in deproteinized biological fluids can be unequivocally reproduced, even if Varying amounts of acid are employed for the deproteinization. For example, up to about 20% trichloroacetic acid has been employed for deproteinization without a substantial difference in the values obtained during the subsequent creatinine determination by said color reagent. Likewise, the determination of creatinine in an acid-free aqueous solution yields approximately the same results as the determination conducted in a solution previously deproteinized by an acid.

Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The following preferred specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the specification and claims in any way whatsoever.

In the examples parts are parts by volume and percentages are in parts by weight per 100 parts by weight unless otherwise indicated. All temperatures are in degrees centigrade.

(A) Examples of color reagents:

EXAMPLE 1 One part of an aqueous 10% lithium hydroxide solution (solution I) is mixed with 5 parts of a picric acid/ lithium hydroxide solution (solution II) produced from 100 ml. of aqueous 1.2% picric acid solution and 1 ml. of solution I).

EXAMPLE 2 One part of an aqueous 9% lithium hydroxide solution is mixed with 5 parts of an aqueous 1.2% picric acid solution.

EXAMPLE 3 One part of a 12% aqueous lithium hydroxide solution is mixed with 5 parts of an aqueous 1.2% picric acid solution.

EXAMPLE 4 One part of a 10% aqueous lithium hydroxide solution is separately mixed with 5 parts of a 0.5% and with 5 parts of a 1.0% aqueous picric acid solution, respectively.

EXAMPLE One part of a aqueous lithium hydroxide solution is separately mixed with 5 parts of an aqueous, weakly acidic 2% and with 5 parts of an aqueous, weakly acidic 3% picric acid solution (prepared by adjusting a 2% and a 3% picric acid solution, respectively, to a weakly acidic pH, e.g. 3 to 7, by means of lithium hydroxide), respectively.

EXAMPLE 6 100 milligrams (mg) of solid picric acid, 167 mg. of solid lithium hydroxide (obtained by evaporating a clear aqueous lithium hydroxide solution) and 100 mg. of lithium chloride are mixed. Before this mixture is utilized, it is dissolved in 10 ml. of distilled water.

EXAMPLE 7 g. of aqueous picric acid 16.7 g. of pure lithium hydroxide and 10 g. of pure lithium chloride are supplemented by distilled water to a volume of 1 liter. The thus-prepared solution is freeze-dried in batches of 0.5 ml., preferably in test tubes.

(B) Examples for creatinine determinations:

EXAMPLE 8 Creatinine determination in aqueous solution 1.0 ml. of the color reagent of Example 1 is mixed with 1.0 ml. of an aqueous solution of creatinine having a pH of 0.2 and is allowed to stand for 10 minutes at 20-25". After several additional minutes (preferably 810 minutes), the extinction of the mixture is measured by means of a spectrophotometer at 520 mp at a cuvette layer thickness of 0.5-2 centimeters (cm.). The creatinine content is determined by comparison with the extinction of a standard solution containing a known quantity of pure creatinine.

Replacing the aqueous creatinine solution by the same volume of aqueous creatinine solutions varying in pH from 0.212, inclusive, but having the same creatinine concentration, leads to the same results. Varying the wavelength at which the extinction is measured to any wavelength from 500 to 550 m e.g. at Hg 546 or Cd 509, also yields accurate data.

EXAMPLE 9 Creatinine determination in a serum (a) Deproteinization:

0.5 ml. of serum is deproteinized in a conventional manner by the addition thereto of 0.5 ml. of a 10% aqueous trichloroacetic acid solution. The mixture is centrifuged for 10 minutes.

For control purposes, 0.5 ml. of a standard solution containing 10 mg. of creatinine per 100 ml. of solution is likewise mixed with 0.5 ml. of a 10% aqueous trichloroacetic acid solution.

Furthermore, a blank sample is produced from 0.5 ml. 10% aqueous trichloroacetic acid solution and 0.5 ml. of water.

(b) Determination of creatinine:

0.5 ml. of the deproteinized serum sample and of the standard and blank samples, respectively, are separately mixed with 0.5 ml. of the color reagent of Example 1 and allowed to stand at 20-25 for 10 minutes. Within another 10 minutes, the extinctions of the serum sample and the standard sample are measured as compared to the blank sample (at a cuvette layer thickness of 0.5-2 cm.) by means of a spectrophotometer between 500 and 550 m preferably at 520 m or, for example, at Hg 546 or Cd 509. The creatinine content is calculated in accordance with the following formula:

Creatinine content in the serum:

E /E -S mg./100 ml.

E =extinction of the serum sample as compared to the blank sample;

E =extinction of the standard sample as compared to the blank sample;

S=concentration of the standard creatinine solution in mg. of creatinine per ml.

If the thus-measured extinction is above 1.0, then the evaluation is advantageously repeated with the sample diluted ten-fold, and the value obtained in accordance with the above formula is then multiplied by 10.

The above-described creatinine determination can be repeated at 15 with the same results.

Conducting the determination with 1 ml. of serum (2.0 ml. of trichloroacetic acid are separately added to 1 ml. of each of serum, standard solution, and water for the blank sample, respectively; and after centrifuging, 1 ml. of residue is mixed with 1 ml. of color reagent) also provides accurate data.

Varying the wavelength at which the extinction is measured to any wavelength from 500 to 550 m e.g. at Hg 546 or Cd 509, also yields accurate data.

Analogous creatinine determinations of serum are likewise conducted with the color reagents of Examples 2 to 7, respectively. When employing lyophilized dosed dry color reagent of Example 7, 1 ml. of 5% trichloroacetic acid is employed during the deproteinization in place of 0.5 ml. of 10% trichloroacetic acid, and 1 ml. of the protein-free residue (as compared to the above-employed 0.5 ml.) is added to the dry color reagent for purposes of the determination.

EXAMPLE 10 Creatinine determination in the urine A creatinine determination is conducted analogously to that of Example 9, with a deproteinized urine sample diluted 100-fold.

The creatinine content is calculated in accordance with the following formula:

Creatinine content of the urine, diluted to 100 times its original volume by means of water:

EA/ES- 1000 mg./100 m1.

E =extinction of the diluted urine sample as compared to the blank sample;

E =extinction of the standard solution as compared to the blank sample.

The normal values in this case are:

1.0-1.5 g./ 24-hour urine 100200 mg./100 ml. of urine.

The preceding examples can be repeated with similar success by substituting the generically or specifically described reactants and/or operating conditions of this invention for those used in the preceding examples.

From the foregoing description, one skilled in the art can easily ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Consequently, such changes and modifications are properly, equitably, and intended to be, within the full range of equivalence of the following claims.

What is claimed is:

1. A composition for the detection of creatinine which consists essentially of lithium hydroxide and at least one member selected from the group consisting of picric acid and lithium picrate, the weight ratio of said lithium hydroxide to said member being 0.3:1 to 0.8:1 respectively, wherein said ratio is calculated on the basis of said member being lithium picrate.

2. A solid color reagent comprising the composition of claim 1 wherein said at least one member is picric 8 acid, and further comprising inert water-soluble solid References Cited diluent to reduce the explosive nature of the picric acid. Clark et a1" AnaL Chem 21 Na 10 October, 1949,

3. A composition according to claim 1 comprising an 1218 1221 intimate admixture of components, said lithium hydroxide being present in an amount sufficient to yield a pH of 5 MORRIS WOLK, primary Examiner 4. A composition according to claim 3, wherein said REEsEAsslstam Exammer composition is a freeze-dried solid. U S Cl X R S. A composition as defined by claim 1 wherein said member is lithiumpicrate. 10 23--230; 260622 6. A composition as defined by claim 5 wherein said ratio is 0.55:1 to 0.65: 1. 

